Affinity Chromatography (AC)

1 Affinity Chromatography (AC)

• Principles of AC

• Main stages in Chromatography

• How to prepare Affinity gel - Ligand Immobilization - Spacer arms – Coupling methods – Coupling tips

• Types of AC

• Elution Conditions

• Binding equilibrium, competitive elution, kinetics

• Industrial Examples: A/G for Therapeutic

• Future Considerations 2 What is affinity chromatography?

Affinity chromatography is a technique of liquid chromatography which separates molecules through biospecific interactions. The molecule to be purified is specifically and reversibly adsorbed to a specific ligand The ligand is immobilized to an insoluble support (“matrix”): resin, “chip”, Elisa plate, membrane, Western, IP (immuneprecipitation), etc Introduction of a “spacer arm” between the ligand and the matrix to improve binding Elution of the bound target molecule a) non specific or b) specific elution method 3 What is it used for?

 Monoclonal and polyclonal

 Fusion proteins

 Enzymes

 DNA-binding proteins ......

 ANY protein where we have a binding partner!! 4 Designing and preparing an affinity gel

 Choosing the matrix

 Designing the ligand - Spacer arms

 Coupling methods

5 Ligand Immobilization

Ligand + Activating agent +

Matrix Activated Immobilised matrix ligand

6 Designing the ligand

 Essential ligand properties: interacts selectively and reversibly with the target

 Carries groups which can couple it to the matrix without losing its binding activity

 Available in a pure form

7 Steric considerations & spacer arms

Small ligand (<1,000) Risk of steric interference with binding between matrix and target molecule

Often need spacer arm

but watch out for Spacer arm adsorption to the spacer!

8 Design of spacer arms

Alkyl chain Real risk of unspecific interactions between H H spacer and target molecule O O

Hydrophilic chain Risk of unspecific interactions greatly O H reduced

No coupling reaction will use 100% of the available binding sites. Using a gel with pre-attached spacer arms will leave some uncoupled spacers, increasing the possibility of non-specific interactions. The unused spacer arms should not be too much of a problem if they are hydrophilic. If available, this problem may be eliminated by using a ligand with a pre-attached spacer arm. 9 Choosing a coupling group

• Ligands are coupled to the matrix via reactive groups

Amino -NH2 Hydroxyl -OH Aldehyde -CHO Thiol -SH Carboxyl -COOH

• Coupling must allow a free binding site

10 Useful Coupling Methods

Coupling chemistry Kinds of group coupled Ligand

N-hydroxysuccinimide -NH2 Protein ,Peptide. Sugar.Polynucleotide.Cofactor

CNBr -NH2 Protein , Sugar.Polynucleotide.Cofactor

Carbodiimide -NH2 Small ligands. Non polar ligand in organic via hydrophilic spacer arm solvents. Derivatized material

Epoxide -SH-NH2-OH Sugars. Small ligands

via hydrophilic spacer arm

Thiol exchange -SH Protein ,Peptide. Polynucleotide Mild Conditions Low MW thiol containig ligand 11 Useful Coupling Methods

Cyanogen bromide activation

O C N O + Gel C N + H Br Gel H Br Sepharose Cyanogen Cyanate ester bromide formed

Cyanogen bromide coupling H O Lig NH O N Gel C N + 2 Gel C Lig

CNBr-activated NH Protein ligand N-substituted isourea Sepharose

Reaction at pH 8. Isourea may be cleaved by nucleophilic attack at high pH. Multiple bonds formed with protein ligands. No spacer arm present. 12 WARNING!: Cyanogen bromide is dangerous. Consult safety data before start working A general protocol for ligand coupling

 Prepare the ligand solution in coupling buffer

 Prepare the activated matrix

 Mix the ligand solution and the activated gel in coupling buffer until coupling is complete

 Check total ligand before and after coupling: ligand/ml resin

 Block any remaining active groups

 Wash the coupled gel alternately at high and low pH to remove adsorbed ligand

 Transfer to storage buffer 13 Tips: Preparing the activated matrix - couple buffer

 If the activated matrix is freeze-dried, allow it to swell before washing

 Always use the recommended swelling and washing solutions!

 The swelling, washing and equilibration are best performed on a sintered glass filter. Facilitates quickly removal of excess liquid from the media

 Couple buffer: make sure there are no components, e.g. amines, that can couple in place of the ligand, e.g. Tris buffer has a primary amine

 To suppress ionic adsorption, add NaCl, 0.5 M.

 pH is important. Always use the recommended pH.

14 Tips: Coupling reaction, washing and storage

 Ligands which are poorly soluble in water, may usually be added in an organic solvent.

 The ligand should be available at a high level of purity. Any contaminants present in the ligand sample will likely be coupled to the matrix.

 Mix the activated gel and the ligand solution by swirling or end-over-end mixing - Avoid magnetic stirrers as these can crush the gel and produce fines!!

 Take samples of ligand before and after reaction: to calculate ligand per ml resin

 Time and temperature according to standard protocols. Most coupling reactions are performed at room temperature

 Block any remaining active groups with a small neutral ligand (Tris buffer for amino reactions)

 Wash the coupled gel at alternating high and low pH to remove adsorbed substances. 15  Keep in storage buffer (Na Azide , Thimerosol 0.02% or ethanol if possible) G Protein Receptor Kinase

Technique Pig brain homogenate Purification Comment factor

Ppt Ammonium sulfate 7 precipitation A. Tobin et al. (1996) J. Biol. Chem. 271, 3907-3916

HIC Butyl Sepharose Fast 20 Flow

AIEX RESOURCE Q

CIEX RESOURCE S 2408

AC HiTrap Heparin 18647 • 10 mg homogenous protein

16 Membrane Protein

Technique Placental extract in Purification Comment 1.5% Triton X-100 factor

AC Blue Sepharose 3 • Concentration and capture

AIEX DEAE Sephacel 4 T. White et al. (1995) J. Biol. Chem. 270, 24156-24165 CIEX SP Sepharose 6

AC Muc2 Sepharose 242 • Main purification step

• Final polishing and purity CIEX Mono S 1442 check, 20 mg obtained

17 DNA-binding Protein

Technique HeLa cell nuclear Purification Comment extract factor J. Berthelsen et al. (1996) J. Biol. Chem. 271, 3822-3830 CIEX SP Sepharose High 5 Performance

AC Heparin Sepharose 8 • General AC step for DNA- Fast Flow binding proteins

AC DNA-1 Sepharose 9 • Removal step, non-specific DNA binding activity removed

AC DNA-2 Sepharose 2447 • Main purification step

CIEX Mono S 4943 • Final polishing, 20 mg obtained

18 Advantages and Disadvantages of affinity chromatography

• Easy to achieve otherwise difficult • Buffer / elution limitations: Sometimes needs harsh or denaturizing separations elution conditions Very expensive elution conditions • Often high purity in one step (but not always) • Economics (scale-up): Ligand stability affects life-time of • Relatively fast separations affinity resins (according to kinetics of binding and elution) Some ligands are very expensive

• Can be use to remove specific • Do not resolve all the problems contaminants

19 The main stages in affinity chromatography

Sample application Elution of the target

Equilibration Wash Re-equilibration Of gel and sample contaminants Of the gel to binding conditions to binding conditions Or keep in storage buffer (20% Ethanol or 0.02%NaAzide in Buffer)

20 Columns and equipment Tips

 Column volume: according to amount of target and gel capacity

 Avoid using excess of resin: to avoid low affinity binding of impurities. Use maximum capacity of the column.

 Column length: not usually critical, short and wide

 Equipment: no special demands

 Equilibrate column before applying sample and follow standard

protocol 21 Sample preparation Tips  Adjust pH, buffer salts and additives to promote binding

 Filter or centrifuge to remove particles

 Make sure that components known to interfere with binding are absent

 Consider alternative capture step before affinity purification

Eliminates crude impurities or interfering substances

IEX or HIC columns are cheaper, with higher dynamic capacity and quickly concentrate lysates

Prolongs resin half life (reduce cost)

22 Sample application Tips

 Binding buffer: usually neutral pH, and high salt concentration when is possible (0.3-0.5M NaCl) to avoid hydrophobic interactions of non specific proteins to the resin. Use additives only if necessary.  Loading directly on column - Strong affinity and fast binding: High flow rate - Weak affinity and/or slow binding: Low flow rate

23 Kinetics of adsorption and desorption Unexpected results  Slow binding

Some targets do not completely bind to the resin: very low dynamic binding capacity

Open columns: slow down binding. Stopped flow binding to bind the target in portions or rebind several times

For batch binding, incubation of around 1.5 hours at 4°C is enough. For extremely slow

binding kinetics, incubate overnight at 4°C. Very gentle stirring

Binding on FPLC column: adjust flow rate to get loading in ~90min

 Slow desorption

If target elutes as a long, low 'peak‘, reduce elution flow-rate Open columns: Stopped flow elution to elute the target in pulses. Change elution scheme

Elution of FPLC column: extremely slow elution in order to get narrow peaks 24 Washing, elution and re equilibration Tips • Follow standard protocol

• Optimize alternative wash strategies to increase final purity (high salt, detergents, others)

• If eluting at extreme pH: collect the target protein in a small amount of concentrated buffer at a neutralizing pH

• Re-equilibrate column immediately with binding or storage buffer

• Regeneration: use buffers that do not harm the ligand 25 Column Regeneration Tips

 Wash with basic and acidic buffers.

– 10 col. vol. 0.1 M Tris.HCl, 0.5 M NaCl, pH 8.5

– 10 col. vol. 0.1 M NaAc, 0.5 M NaCl, pH 4.5

– Use chaotropic agents or detergents if needed

 Re-equilibrate with 10 column volumes starting buffer. Use more if needed

 Ni columns can be washed with 0.5N NaOH, neutralized, destriped with

100mM neutral EDTA, wash, charge with 100mM Ni SO4, wash and store with 20% Ethanol 26 Column Storage Tips

 To maximize the useful lifetime of an affinity resin, choose a storage buffer that does not harm the ligand

 Typical buffers are 20% ethanol solution or binding buffer containing a bacteriostatic agent (e.g. 0.02% Thimerosal or 0.02% sodium azide)

 Resins with strong and stable ligands can be used hundreds of

times 27 Affinity Chromatography (AC)

• Principles of AC

• Main stages in Chromatography

• How to prepare Affinity gel - Ligand Immobilization - Spacer arms – Coupling methods – Coupling tips

• Types of AC

• Elution Conditions

• Binding equilibrium, competitive elution, kinetics

• Industrial Examples: Protein A/G for Therapeutic proteins

• Future Considerations 28 Type of Affinities

Mono-specific ligands Group-specific ligands

• Specific for a single substance • Specific for a group of structurally or functionally similar substances: Lectins glycoproteins Hormone receptor Protein G IgG antibodies • Usually home-made gels Dye-stuffs enzymes • Elution scheme must be worked • Often ready-made gels out for each case: • Known elution schemes • Often general elution • Standard tested elution protocols • Little help from the literature • Often competitive elution 29 Group-specific ligands - 1

Ligand Specificity

Protein A Fc region of IgG

Protein G Fc region of IgG

Concanavalin A Glucopyranosyl & Mannopyranosyl groups

Peanut Lectin Terminal galactose group

Cibacron Blue Broad range of enzymes, serum albumin

Procion Red NADP+ dependent enzymes

Lysine Plasminogen, ribosomal RNA 30 Group-specific ligands - 2

Ligand Specificity

Arginine Serine proteases

Benzamidine Serine proteases

Calmodulin Proteins regulated by calmodulin

Heparin Coagulation factors, lipoproteins, lipases,

hormones, steroid receptors, protein synthesis factors, Nucleic acid-binding enzymes Metal chelate resins Proteins and peptides which contain 6-12 His (Co, Ni, Zn, Cu, Fe) 31 Designing the elution scheme

 General elution conditions

– Low pH, salt, GuHCl, Urea etc.

– Often harsh conditions

 Specific eluents

– Competing free ligand

– Competing binding substances

– Sometimes expensive compounds (like peptides) 32 General elution conditions

+

Changing buffer conditions Usually decrease pH or increase ionic strength

decrease polarity adding up to 10 % dioxane or up to 50 % ethylene glycol

Reconstituting + buffer Denaturing buffer Usually extremes of pH or chaotropic agents

There are no guaranteed general elution methods in affinity chromatography

33  Low pH, e.g. glycine HCl, pH 2.8, is the closest approach General elution conditions • pH • 100 mM glycine•HCl, pH 2.5-3.0 • 100 mM citric acid, pH 3.0 Elution conditions are intended to break • 50-100 mM triethylamine or triethanolamine, pH 11.5 • 150 mM ammonium hydroxide, pH 10.5 the ionic, hydrophobic and hydrogen bonds that hold the ligand and the target together • Ionic strength and/or chaotropic effects • 3.5-4.0 M magnesium chloride, pH 7.0 in 10 mM Tris  Successful eluting conditions will be • 5 M lithium chloride in 10 mM phosphate buffer, pH 7.2 • 3.0 M Potassium chloride dependent upon the specific ligand-target • 2.5 M sodium iodide, pH 7.5 • 0.2-3.0 sodium thiocyanate interaction that is occurring.

• Denaturing Ideally, an elution condition effectively • 2-6 M guanidine•HCl • 2-8 M urea releases the target without causing • 1% deoxycholate permanent damage, but all eluting • 1 % SDS conditions result in some loss of functionality • Organic • 10% dioxane Empirical evidence is needed to determine • 50% ethylene glycol, pH 8-11.5 (also chaotropic) which elution condition is the best. • Competitor 34 • >0.1 M counter ligand or analog Elution at Low pH IgG antibodies on rProtein A Sepharose

 Column: HiTrap rProtein A

 Binding: 20 mM sodium phosphate, pH 7.0

 Elution: 0.1 M glycine HCl, pH 3

Check for stabilizing elution conditions: slightly higher pH, stabilizing additives as Arginine, kosmotropic salts, etc

Note: Collect IgG into a small volume of strong buffer (1M pH 7.5) , to preserve its antibody activity. 35 Elution at High salt concentration DNA-binding proteins on Heparin Sepharose

 Column: HiTrap Heparin

 Binding: 20 mM Tris-HCl, pH 8, 0.5 M NaCl

 Elution: Binding buffer + 1 to 2 M NaCl

Heparin Sepharose behavior is similar to CEIX Consider use of CEIX 36 Specific eluents

+

Competing ligand in solution Elution of enzymes from Blue Sepharose by free NADH Glutathione elution of GST fusion protein from Glutathione-Agarose columns

+

Competing binding substance in solution Elution of glycoproteins from Con A Sepharose by a-D-methylmannoside Elution of 6His-Proteins from chelating columns (Ni-NTA) with Imidazole Elution of from Antibody columns with specific peptides  Competitive soluble ligands or binding substances can elute the bound target specifically  Are usually far more gentle than general methods  But sometimes could be very expensive (like specific peptides for antigen elution) 37 Lectins

Lectins are proteins Lectin Specificity which bind well-defined Concanavalin A α-mannose, α-glucose Glycine Max (soybean) N-Acetyl Galactosamine sugar residues of Ulex Europaeus I α-L-fucose polysaccharides, Wheat germ lectin N-Acetyl Glucosamine & NeuNAc glycoproteins, etc Peanut lectin α-mannose

Ricinus Communis, RCA 120 ß-Galactose Elution with competing Arachis hypogaea ß-Gal(1->3)GalNAc free binding substance: Bandeira Simplicifolia, BS-I α-Gal & α Gal NAc Maackia amurensis Sialic acid sugar Lymulus polyphemus NeuNAc 38 Dye-stuffs

Many dye-stuffs mimic binding sites of natural biologically active molecules Elution with High Salt

Dye-stuff Sites recognised Cibacron Blue 3G-A NADH-binding and similar Procion Red NADPH-binding and similar

The structural analogies are not exact. Related structures are also recognized.

Blue Sepharose also binds serum albumin. 39 Binding equilibrium

At equilibrium  LT KD   LT

KD is the equilibrium dissociation constant, [L] is the concentration of free ligand [T] is the concentration of free target [LT] is the concentration of the ligand/target complex

40 Binding equilibrium

It can be shown* that Bound target  L0 Total target K D  L0

KD is the equilibrium dissociation constant, -4 -2 L0 is the concentration of ligand, usually 10 - 10 M

For good binding, KD should be at least two orders of -6 -4 magnitude less than L0, i.e. 10 - 10 M

*Graves, DJ, Wu, YT Meth. Enzymol 34 (1974) 140-163 41 Binding equilibria

We can change KD by changing pH, temperature, salt concentration etc.

Binding

-6 -4 L + T LT KD 10 - 10 M

-1 -2 L + T LT KD 10 - 10 M Elution

Target elutes as a sharp peak

42 Elution by changing KD - Unexpected results

 KD too high during binding (low affinity) Target is retarded as a broad peak and elutes under binding conditions as a broad, low peak Find better binding conditions or use ligand with higher affinity

 KD not low enough during elution Target elutes in a long, low 'peak'

Try different elution conditions or use ligand with lower affinity

 KD too low during binding (extremely high affinity)

Difficult to elute target. Difficult or impossible to increase KD enough to elute the target without destroying its activity 43 Use ligand with lower affinity Competitive elution

Binding + Release

Elution +

C + T CT

44 Binding equilibrium for competing ligand

At equilibrium CT KDComp  CT

KDComp is the equilibrium dissociation constant [C] is the concentration of free competing ligand [T] is the concentration of free target [CT] is the concentration of the competing ligand/target complex

45 Affinity Chromatography (AC)

• Principles of AC

• Main stages in Chromatography

• How to prepare Affinity gel - Ligand Immobilization - Spacer arms – Coupling methods – Coupling tips

• Types of AC

• Elution Conditions

• Binding equilibrium, competitive elution, kinetics

• Industrial Examples: Protein A/G for Therapeutic proteins

• Future Considerations 46 Antibody’s regions

47 Enzymatic cleavage of Antibody Recombinant Ab fragments production

Various modifications of both native and recombinant antibodies are possible Affinity ligands for antibody purification

49 Protein G and protein A bind to different IgG through the Fc region

Protein G and protein A are bacterial proteins from Group G Streptococci and Staphylococcus aureus, respectively Overview of different combinations of chromatography unit operations in a MAb purification process

Removal of specific contaminants after initial purification

• Bovine immunoglobulins • Albumin and transferrin • α2 -macroglobulin and • haptoglobulin • DNA and endotoxins • Dimers and aggregates • Host cell proteins (HCP) • Virus Dynamic binding capacity of MAb at various residence times

52 Protein L binds to the variable region of the kappa light chain

It can be used to purify intact immunoglobulins conventional Fabs chain variable fragments (scFvs)  Was first isolated from the surface of bacterial species Peptostreptococcus magnus

Immunoglobulins whose Fc region is not easily accessible for

Protein A, such as IgM or IgA. 53 Protein L binding affinities De Chateau, M. et al. On the interaction between protein L and immunoglobulins of various mammalian species. Scand. J. Immunol. 37, 399-405 (1993).

54 Design of a new Affinity Chromatography Parameters to consider

• Ligand that binds to the resin covalently and keeps the interaction site free • Low leakiness of the ligand • Stability of the ligand to proteases, and to classical regeneration conditions: 0.5N NaOH treatment, urea or GuHCl • High specificity of the ligand • But easy release of the target by non-denaturative conditions • Cheap elution conditions

• Cheap production of the ligand 55 Single domain antibodies (sdAb) Five major classes of Immunoglobulin Advantages of target specific sdAb www.GenScript.com

Conventional IgGs consist • sdAb) retain all the function of conventional IgGs despite of two heavy chains and two light lacking the Vl binding region chains. Variable regions o both chains • Use only the VH H fragment for binding contribute to antigen binding. • Benefits: 1/10 the size of convention IgGs, ~13 kDa vs ~150 kDa Camelids produce antibodies • Readily amenable to downstream engineering such as composed of only two heavy humanization chains, called heavy chain • Access to hidden epitopes antibody (HCAb). • Better penetration into solid tissue • Ability to cross the blood brain barrier Single domain antibodies (sdAb) • High pH and temperature stability: consist of only the variable region of • Permits alternate routes of administration (i.e. oral) HCAb, since it is the sole region • Use in other extreme environments (biosensors) responsible for binding. These are • Use as a ligand for affinity resins called VHH fragments Workflow for generating an sdAb target specific www.GenScript.com

Immunization Isolation from Naïve sdAb of llamas Library

Collect blood and isolate leukocytes

Detection of heavy chain Ab response sdAb sequencing, subcloning and expression Cloning of sdAb DNA Panning against antigen into Phage Display and phage ELISA on vector individual clones Affinity and Biophysical Optimization Camelid derived single domain antibody fragments BAC (BioAffinity Company) - Capture Select® (GE)

 Custom designed ligands (12-15kDa) •Capture Select human IgA  Produced in Saccharomyces cerevisiae, enabling •CaptureSelect IgM directed towards a high level production with minimal purification unique domain on the Fc part of IgM effort as well as easy scale-up. •CaptureSelect Fab kappa: enable  Stability, affinity, and selectivity purification of human IgG, IgA, IgM, and IgE.  Reduction of process steps: higher yields, reduced costs •CaptureSelect Fab lambda: enable  Selectivity: high purity in single step / feed stock purification of human IgG, IgA, IgM, and IgE. independent •CaptureSelect Human IgG4 : enables  Mild elution conditions: retaining biological purification of human IgG4’s without cross activity of target reactivity with human IgG from subclasses 1,  Efficient clearance of HCP, DNA, virus: high 59 selectivity in capture step 2, and 3 (Designed Ankyrin Repeat Proteins) An Innovative High-Throughput Platform for Next-Generation Binder Discovery University of Zurich, Switzerland - Prof. Dr. Andreas Plückthun – Dr. Jonas Schaefer

Designed Ankyrin Repeat Proteins (DARPins) against different epitopes on a variety of targets Ankyrin repeat is a 33 residue motif • small (~170 aa), very stable repeat proteins in proteins consisting of two alpha helices separated by loops • recognize structural 3D-epitopes Domains consisting of ankyrin repeats mediate • work intracellularly (no Cys) protein-protein interactions and are among the most • available in flexible formats / modifications common structural motifs in known proteins Provide monomeric binders that specifically recognize different, non-overlapping epitopes with high affinities HT Ribosome Display (RD) enables selection against 94 targets in parallel High-Throughput Screenings allow to identify best suited DARPin binders

Plückthun, A. (2015) DARPins: Binding Proteins for Research, Diagnostics, and Therapy. Annu Rev Pharmacol Toxicol. 555:489-511 Mimetic Ligand™ Affinity Chromatography Synthetically “mimicking” and enhancing the natural molecular affinity of binding ligands toward targeted biomolecules www.prometic.com

• Biologically inert • very strong • excellent liquid flow properties • resistant to most chemical treatments • stable in the pH range 2 - 14 • can be treated with denaturants (8M urea), detergents (Triton, SDS) • can be sterilized by autoclaving.  Capacities can exceed 50mg protein per ml of packed gel. Low Ligand Leakage

 Alkali-stable adsorbents - Enables cleaning, sterilization and depyrogenation with 1M sodium hydroxide, ensuring long operational lifetimes.

 Albumin • Proteases • Blood Proteins • Oxidases • Dehydrogenases • Ligases • Kinases • Antibodies • Nucleases •

 Custom Media Development Programs

 Chemical Combinatorial Library™, + with a rational ligand design approach 61 Scil Proteins: Affilin™ Techology

 Novel human binding proteins for therapy and applications in chromatography.

 Affilin™ molecules are small non-immunoglobulin proteins which are designed for specific binding of proteins and small molecules.

 New Affilin™ molecules can be quickly selected from libraries which are based on two different human derived scaffold proteins: Gamma crystalline, a human structural eye lens protein and one of the highest conserved proteins known

• Small scaffolds

• High temperature stability

• Almost resistant to pH changes and denaturing agents.

 Created by engineering the protein surfaces through a locally defined randomization of exposed amino acids

 Can be easily produced in the cytoplasm of E. coli 62 Non-immunoglobulin63 scaffolds: a focus on their targets Katja Skrlec et al. Trends in Biotechnology July 2015, Vol. 33, No. 7 64 Charge-Variant Profiling of Biopharmaceuticals

LCGC NORTH AMERICA VOLUME 36 NUMBER 1 JANUARY 2018 Jared R. Auclair, Anurag Rathore, and Ira Krull

Y. Du, A. Walsh, R. Ehrick, W. Xu, K. May, and H. Liu, mAbs 4(5), 578–585 (2012).

The term acidic species refers to a negatively charged species; using cation exchange these variants are eluted before the main peak, and when using anion exchange they are eluted after the main peak

The term basic species refers to positively charged species; using cation exchange HPLC, these variants are eluted after the main peak, and when using anion exchange these variants are eluted before the main peak. Acidic species and Basic species Y. Du, A. Walsh, R. Ehrick, W. Xu, K. May, and H. Liu, mAbs 4(5), 578–585 (2012)

• Sialic acid • C-terminal Lys • Deamidation • N-terminal Glu • Nonclassical linkage • Isomerization of Asp • Trisulfide bonds • Succinimide • High mannose • Met oxidation • Thiosulfide modification • Amidation • Glycation • Incomplete disulfide bonds • Modification by maleuric acid • Incomplete removal of leader sequence • Cysteinylation • Mutation from Ser to Arg • Reduced disulfide bonds • Aglycosylation • Nonreduced species • Fragments • Fragments • Aggregates Bispecific Antibodies Bispecific Antibodies Close in on CancerMarianne Guenot, GEN Exclusives February 27, 2017 • Bispecific antibodies are products of genetic engineering that allow one antibody-like molecule to bind several different antigens at once • In December 2014, for the first time, a bispecific antibody, blinatumomab (Blincyto©), was approved for therapeutic use. • Amgen’s blinatumomab, is a molecule composed of the single-chain variable region (scFv) of one antibody targeting the T-cell activating molecule CD3, linked to another scFv, which binds to the B-cell antigen CD19. In the context of acute lymphoblastic leukemia (ALL), this bispecific antibody brings the T cell’s potent cytotoxicity close to the malignant B cells, allowing them to be specifically targeted. • Since then, over 120 bispecific molecules have entered the clinical pipeline • Global market for 2024 is estimated to hit a staggering $5.8 billion annually, Research and Markets report (“Bispecific Antibody Therapeutics Market, 2014–2024”). • Can bring clinical benefits beyond the injection of two monospecific antibodies 67 What are Biosimilars? Biosimilars. Are They Ready for Primetime? Gina Battaglia and Leala Thomas — July 12, 2017 – Bioradiations BIORAD http://www.bioradiations.com/biosimilars-are-they-ready-for- primetime/?WT.mc_id=170712020560&mkt_tok=eyJpIjoiTUdSaE1XUXpPVE16TVdZMCIsInQiOiJ1VTh4d2RJT1wvcHY5Z0tSckdXQ201VkFmdkUySHZQUGhEY1BoT1A5YVNsbTRBQXp mZk1ZdFVSZ09xUVNadmV6S1EyNE1sUnVvamlEU1JYUEY4MmMxaWlxQUowc1l1VndNQ1hcLzhMdzRWMFZRNkdPbUdKbzROcWFGbE13S05mTmJEIn0%3D#references

 A biosimilar is “a product that is highly similar to the reference product despite minor differences in clinically inactive components; and there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product,”

Emanuela Lacana, PhD, Associate Director, Biosimilars and Biologics Policy, Office of Biological Products, CDER, FDA, at the PDA/FDA Joint Regulatory Conference in Washington, DC in September 2016 (Tierney 2016).  More simply, it is a biologic that is almost identical to a previously approved biological product, with no clinically meaningful differences in safety or efficacy.

 Market is expected to grow to US$11 billion by 2021 from US$3.4 billion in 2016

 Key players in the biosimilars market include by Pfizer Inc. (U.S.), Sandoz International GmbH (Germany), Teva Pharmaceuticals Industries Ltd. (Israel), Amgen Inc. (U.S.), Biocon Ltd. (India), Dr. Reddy’s Laboratories Ltd. (India), F. Hoffmann-La Roche Ltd. (Switzerland), Celltrion, Inc. (South Korea), and Samsung Bioepis (South Korea).

Currently account for 20% of the global pharmaceutical market. $2.6 billion to develop a new biologic, and development time of over a decade (Di Masi et al.2016).

The European Union approved its first biosimilar in 2006 and leads the global biosimilar market with 35 approved products. In addition, they were the first to develop product-specific development guidelines

Biosimilar development requires extensive physicochemical and biological characterization of the reference originator product using multiple analytical techniques to ensure similarity. Based on the results of this characterization, preclinical studies and clinical trials are undertaken to resolve uncertainty about the biosimilarity of the new biosimilar product.

The uncertainty about legal issues (for example, patent infringement) and the complex expertise required to synthesize biologics will likely mean that biosimilar development will be limited to large pharmaceutical companies with secure finances and extensive experience.